Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Relationship between saxagliptin use and left ventricular diastolic function assessed by cardiac MRI.

AIMS: Type 2 diabetes mellitus (T2DM) increases the risk of major cardiovascular events. In SAVOR-TIMI53 trial, the excess heart failure (HF) hospitalization among patients with T2DM in the saxagliptin group remains poorly understood. Our aim was to evaluate left ventricular (LV) diastolic function after 6 months of saxagliptin treatment using cardiac magnetic resonance imaging (CMR) in patients with T2DM. METHODS: In this prospective study, 16 T2DM patients without HF were prescribed saxagliptin as part of routine guideline-directed management. CMR performed at baseline and 6 months after initiation of saxagliptin treatment were evaluated in a blinded fashion. We assessed LV diastolic function by measuring LV peak filling rate with correction for end-diastolic volume (PFR/LVEDV), time to peak filling rate with correction for cardiac cycle (TPF/RR), and early diastolic strain rate parameters [global longitudinal diastolic strain rate (GLSR-E), global circumferential diastolic strain rate (GCSR-E)] by feature tracking (FT-CMR). RESULTS: Among the 16 patients (mean age of 59.9, 69% males, mean hemoglobin A1c 8.3%, mean left ventricular ejection fraction 57%), mean PFR was 314 ± 108 ml/s at baseline and did not change over 6 months (- 2.7, 95% CI - 35.6, 30.2, p = 0.86). There were also no significant changes in other diastolic parameters including PFR/EDV, TPF, TPF/RR, and GLSR-E and GCSR-E (all p > 0.50). CONCLUSION: In T2DM patients without HF receiving saxagliptin over 6 months, there were no significant subclinical changes in LV diastolic function as assessed by CMR.

Prognostic Value of Different Thresholds for Myocardial Scar Quantification on Cardiac MRI Late Gadolinium Enhancement Images in Patients Receiving Implantable Cardioverter Defibrillators.

Purpose: To compare the predictive value of different myocardial scar quantification thresholds using cardiac MRI for appropriate implantable cardioverter defibrillator (ICD) shock and mortality. Materials and Methods: In this retrospective, two-center observational cohort study, patients with ischemic or nonischemic cardiomyopathy underwent cardiac MRI prior to ICD implantation. Late gadolinium enhancement (LGE) was first determined visually and then quantified by blinded cardiac MRI readers using different SDs above the mean signal of normal myocardium, full-width half-maximum, and manual thresholding. The intermediate signal "gray zone" was calculated as the differences between different SDs. Results: Among 374 consecutive eligible patients (mean age, 61 years ± 13 [SD]; mean left ventricular ejection fraction, 32% ± 14; secondary prevention, 62.7%), those with LGE had a higher rate of appropriate ICD shock or death than those without (37.5% vs 26.6%, log-rank P = .04) over a median follow-up of 61 months. In multivariable analysis, none of the thresholds for quantifying scar was a significant predictor of mortality or appropriate ICD shock, while the extent of gray zone was an independent predictor (adjusted hazard ratio per 1 g = 1.025; 95% CI: 1.008, 1.043; P = .005) regardless of the presence or absence of ischemic heart disease (P interaction = .57). Model discrimination was highest for the model incorporating the gray zone (between 2 SD and 4 SD). Conclusion: Presence of LGE was associated with a higher rate of appropriate ICD shock or death. Although none of the scar quantification techniques predicted outcomes, the gray zone both in infarct and nonischemic scar was an independent predictor and may refine risk stratification.Keywords: MRI, Scar Quantification, Implantable Cardioverter Defibrillator, Sudden Cardiac Death Supplemental material is available for this article. © RSNA, 2023.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Evaluation of left atrial remodeling using cardiovascular magnetic resonance imaging in breast cancer patients treated with adjuvant trastuzumab.

OBJECTIVES: We evaluated left atrial (LA) remodeling using cardiac MRI (CMR) in patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer during and after trastuzumab therapy. METHODS: In this prospective 2-center longitudinal study, 41 women with HER2-positive breast cancer received adjuvant trastuzumab for 12 months, in addition to standard chemotherapy. Serial CMRs were performed at baseline, 6, 12, and 18 months after initiation of trastuzumab. LA volumes were measured by a blinded reader. Linear mixed model was used to evaluate longitudinal changes. RESULTS: Of 41 women (mean age 52 ± 11 [SD] years; 56% received anthracycline), one patient experienced trastuzumab-induced cardiotoxicity (TIC) for which trastuzumab was interrupted for one cycle. Mean baseline left ventricular ejection fraction (LVEF) was 68.0 ± 5.9% and LA ejection fraction (LAEF) was 66.0 ± 6.6%. Compared to baseline, LAEF decreased significantly at 6 months (62.7 ± 5.7%, p = 0.027) and 12 months (62.2 ± 6.1%, p = 0.003), while indexed LA minimum volume (LAmin) significantly increased at 12 months (11.6 ± 4.9 ml/m2 vs 13.8 ± 4.5 ml/m2, p = 0.002). At 18 months, all changes from baseline were no longer significant. From baseline to 6 months, change in LAEF correlated with change in LVEF (Spearman's r = 0.41, p = 0.014). No significant interactions (all p > 0.10) were detected between time and anthracycline use for LA parameters. CONCLUSIONS: Among trastuzumab-treated patients with low incidence of TIC, we observed a small but significant decline in LAEF and increase in LAmin that persisted for the duration of therapy and recovered 6 months after therapy cessation. These findings suggest that trastuzumab has concurrent detrimental effects on atrial and ventricular remodeling. KEY POINTS: • In trastuzumab-treated breast cancer patients evaluated by cardiac MRI, left atrial ejection fraction declined and minimum volume increased during treatment and recovered to baseline after trastuzumab cessation. • Changes in left atrial ejection fraction correlated with changes in left ventricular ejection fraction in the first 6 months of trastuzumab treatment. • Trastuzumab therapy is associated with concurrent detrimental effects on left atrial and ventricular remodeling.

The Learning, Social and Emotion Adaptation Questionnaire-Short Form: A Measure of Adaptive Behavior for Primary School Students with Autism Spectrum Disorder.

Students with autism spectrum disorder (ASD) studying in mainstream classrooms have diverse adjustment difficulties in learning, social interaction, and emotion regulation. It is crucial to identify the areas these students find most challenging so that teachers can provide training and support accordingly. We therefore developed, examined, and provided norms for the Learning, Social and Emotion Adaptation Questionnaire-Short Form (LSEAQ-S), a teacher report instrument measuring 53 essential adaptive behaviors for mainstream primary school students in Hong Kong. Teachers completed the LSEAQ-S for three samples of 2,298, 2,690, and 3,305 students with ASD from 204 schools and a sample of 1,869 students without ASD from 112 schools. Our study showed that an 11-factor structure best describes the LSEAQ-S, which has high internal consistency and good convergent validity examined with the Social Responsiveness Scale-Second Edition (SRS-2). Normative data of the LSEAQ-S stratified by gender and grade (grades 1 to 3; grades 4 to 6) are presented. Gender and grade differences were found, with girls with ASD lagging behind their same-gender peers in related skills more than boys with ASD did, across both grade levels and especially in senior grades. The LSEAQ-S, together with its normative data, can reveal students' difficulties and needs, inform intervention priorities, and help monitor training progress. LAY SUMMARY: This study introduces the Learning, Social and Emotion Adaptation Questionnaire-Short Form (LSEAQ-S), a teacher report instrument developed in Hong Kong measuring school adaptation of students with autism spectrum disorder (ASD) in mainstream primary schools. The measure helps education personnel identify behaviors in which a student falls behind his/her peers and facilitate training and support targeting those behaviors. Autism Res 2021, 14: 959-972. © 2020 The Authors. Autism Research published by International Society for Autism Research and Wiley Periodicals LLC.

Ankyrin repeats as a dimerization module.

Legionella pneumophila is a pathogen, causing severe pneumonia in humans called Legionnaires' disease. AnkC (LegA12) is a poorly characterized 495-residue effector protein conserved in multiple Legionella species. Here, we report the crystal structure of a C-terminally truncated AnkC (2-384) at 3.2 Å resolution. The structure shows seven ankyrin repeats (ARs) with unique structural features. AnkC forms a dimer along the outer surface of loops between ARs. The dimer exists both in the crystal form and in solution, as shown by analytical ultracentrifugation. This is the first example of ARs as a dimerization module as opposed to solely a protein interaction domain. In addition, a novel α-helix insert between AR3-AR4 is positioned across the surface opposite the ankyrin groove. Sequence conservation suggests that the ankyrin groove of AnkC is a functional site that interacts with binding targets. This ankyrin domain structure is an important step towards a functional characterization of AnkC.

Crystal structure of the Legionella effector Lem22.

Legionella pneumophila is a pathogen causing severe pneumonia in humans called Legionnaires' disease. Lem22 is a previously uncharacterized effector protein conserved in multiple Legionella strains. Here, we report the crystal structure of Lem22 from the Philadelphia strain, also known as lpg2328, at 1.40 Å resolution. The structure shows an up-and-down three-helical bundle with a significant structural similarity to a number of protein-binding domains involved in apoptosis and membrane trafficking. Sequence conservation identifies a putative functional site on the interface of helices 2 and 3. The structure is an important step toward a functional characterization of Lem22.

Safety of Active Rehabilitation for Persistent Symptoms After Pediatric Sport-Related Concussion: A Randomized Controlled Trial.

OBJECTIVES: To examine the safety and tolerability of an active rehabilitation program for adolescents who are slow to recover from a sport-related concussion, and secondarily to estimate the treatment effect for this intervention. DESIGN: Single-site, parallel, open-label, randomized controlled trial comparing treatment as usual (TAU) to TAU plus active rehabilitation. SETTING: Outpatient concussion clinic. PARTICIPANTS: Adolescents (N=19) aged 12 to 18 years with postconcussion symptoms lasting ≥1 month after a sports-related concussion. INTERVENTIONS: TAU consisted of symptom management and return-to-play advice, return-to-school facilitation, and physiatry consultation. The active rehabilitation program involved in-clinic subsymptom threshold aerobic training, coordination exercises, and visualization and imagery techniques with a physiotherapist (mean, 3.4 sessions) as well as a home exercise program, over 6 weeks. MAIN OUTCOME MEASURES: A blinded assessor systematically monitored for predetermined adverse events in weekly telephone calls over the 6-week intervention period. The treating physiotherapist also recorded in-clinic symptom exacerbations during aerobic training. The Post-Concussion Symptom Scale was the primary efficacy outcome. RESULTS: Nineteen participants were randomized, and none dropped out of the study. Of the 12 adverse events detected (6 in each group), 10 were symptom exacerbations from 1 weekly telephone assessment to the next, and 2 were emergency department visits. Four adverse events were referred to an external safety committee and deemed unrelated to the study procedures. In-clinic symptom exacerbations occurred in 30% (9/30) of aerobic training sessions, but resolved within 24 hours in all instances. In linear mixed modeling, active rehabilitation was associated with a greater reduction on the Post-Concussion Symptom Scale than TAU only. CONCLUSIONS: The results support the safety, tolerability, and potential efficacy of active rehabilitation for adolescents with persistent postconcussion symptoms.

Divergent evolution of Di-lysine ER retention vs. farnesylation motif-mediated anchoring of the AnkB virulence effector to the Legionella-containing vacuolar membrane.

Within macrophages and amoeba, the Legionella-containing vacuole (LCV) membrane is derived from the ER. The bona fide F-box AnkB effector protein of L. pneumophila strain AA100/130b is anchored to the cytosolic side of the LCV membrane through host-mediated farnesylation of its C-terminal eukaryotic "CaaX" motif. Here we show that the AnkB homologue of the Paris strain has a frame shift mutation that led to a loss of the CaaX motif and a concurrent generation of a unique C-terminal KNKYAP motif, which resembles the eukaryotic di-lysine ER-retention motif (KxKxx). Our phylogenetic analyses indicate that environmental isolates of L. pneumophila have a potential positive selection for the ER-retention KNKYAP motif. The AnkB-Paris effector is localized to the LCV membrane most likely through the ER-retention motif. Its ectopic expression in HEK293T cells localizes it to the perinuclear ER region and it trans-rescues the ankB mutant of strain AA100/130b in intra-vacuolar replication. The di-lysine ER retention motif of AnkB-Paris is indispensable for function; most likely as an ER retention motif that enables anchoring to the ER-derived LCV membrane. Our findings show divergent evolution of the ankB allele in exploiting either host farnesylation or the ER retention motif to be anchored into the LCV membrane.

Structural Mimicry by a Bacterial F Box Effector Hijacks the Host Ubiquitin-Proteasome System.

Ankyrin B (AnkB/LegAU13) is a translocated F box effector essential for the intracellular replication of the pathogen Legionella pneumophila. AnkB co-opts a host ubiquitin ligase to decorate the pathogen-containing vacuole with K48-linked polyubiquitinated proteins and degrade host proteins as a source of energy. Here, we report that AnkB commandeers the host ubiquitin-proteasome system through mimicry of two eukaryotic protein domains. Using X-ray crystallography, we determined the 3D structure of AnkB in complex with Skp1, a component of the human SCF ubiquitination ligase. The structure confirms that AnkB contains an N-terminal F box similar to Skp2 and a C-terminal substrate-binding domain similar to eukaryotic ankyrin repeats. We identified crucial amino acids in the substrate-binding domain of AnkB and showed them to be essential for the function of AnkB in L. pneumophila intracellular proliferation. The study reveals how Legionella uses molecular mimicry to manipulate the host ubiquitination pathway and proliferate intracellularly.

Crystal Structure of the Salmonella Typhimurium Effector GtgE.

Salmonella Typhimurium GtgE is an effector protein contributing to the virulence of this pathogen. It was shown to possess highly selective proteolytic activity against a subset of Rab proteins that helps in evasion of Salmonella-containing vacuole (SCV) fusion with lysosomes. Cys45, His151 and Asp169 are essential for proteolytic activity. The structure of a C-terminal fragment GtgE(79-214) indicated the presence of a papain-like fold. Here, we present the structure of GtgE(17-214) containing the fully assembled active site. The design of a proteolytically active and crystallizable GtgE construct was aided by NMR spectroscopy. The protein indeed displays papain-like fold with an assembled Cys-His-Asp catalytic triad. Like the full-length GtgE, the crystallizable construct showed low activity in vitro for its known substrates, Rab32 and Rab29. NMR titration experiments showed at most very weak binding of GtgE to the peptide encompassing the Rab29 cleavage site. In view of the low in vitro activity and poor substrate binding, we postulate that the function of GtgE in vivo as a proteolytic enzyme is dependent on other factor(s), such as a protein partner or interactions with the SCV membrane, which stimulate(s) GtgE activity in vivo.

Structure of the Legionella Effector, lpg1496, Suggests a Role in Nucleotide Metabolism.

Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaires disease. Here, we crystallized three nucleotide binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term the KLAMP domain due to the presence of homologous domains in bacterial histidine kinase-like ATP binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3',5'-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3',5'-cAMP reveals the contribution of Tyr-61 and Tyr-69 that produces π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide binding domains associated with bacterial virulence.

High-throughput capturing and characterization of mutations in essential genes of Caenorhabditis elegans.

BACKGROUND: Essential genes are critical for the development of all organisms and are associated with many human diseases. These genes have been a difficult category to study prior to the availability of balanced lethal strains. Despite the power of targeted mutagenesis, there are limitations in identifying mutations in essential genes. In this paper, we describe the identification of coding regions for essential genes mutated using forward genetic screens in Caenorhabditis elegans. The lethal mutations described here were isolated and maintained by a wild-type allele on a rescuing duplication. RESULTS: We applied whole genome sequencing to identify the causative molecular lesion resulting in lethality in existing C. elegans mutant strains. These strains are balanced and can be easily maintained for subsequent characterization. Our method can be effectively used to analyze mutations in a large number of essential genes. We describe here the identification of 64 essential genes in a region of chromosome I covered by the duplication sDp2. Of these, 42 are nonsense mutations, six are splice signal mutations, one deletion, and 15 are non-synonymous mutations. Many of the essential genes in this region function in cell cycle, transcriptional regulation, and RNA processing. CONCLUSIONS: The essential genes identified here are represented by mutant strains, many of which have more than one mutant allele. The genetic resource can be utilized to further our understanding of essential gene function and will be applicable to the study of C. elegans development, conserved cellular function, and ultimately lead to improved human health.

Structure of parkin reveals mechanisms for ubiquitin ligase activation.

Mutations in the PARK2 (parkin) gene are responsible for an autosomal recessive form of Parkinson's disease. The parkin protein is a RING-in-between-RING E3 ubiquitin ligase that exhibits low basal activity. We describe the crystal structure of full-length rat parkin. The structure shows parkin in an autoinhibited state and provides insight into how it is activated. RING0 occludes the ubiquitin acceptor site Cys(431) in RING2, whereas a repressor element of parkin binds RING1 and blocks its E2-binding site. Mutations that disrupted these inhibitory interactions activated parkin both in vitro and in cells. Parkin is neuroprotective, and these findings may provide a structural and mechanistic framework for enhancing parkin activity.

Comparative hemodynamic effects of levosimendan alone and in conjunction with 4-aminopyridine or calcium chloride in a rodent model of severe verapamil poisoning.

Levosimendan (Levo) increases sensitivity of troponin-C to calcium, thus increasing myocardial contractility. It is also a vascular K+-ATP channel agonist producing peripheral vasodilation. Previous research with levosimendan revealed an increase in cardiac output (CO) but not blood pressure (BP) in experimental verapamil poisoning. Levosimendan's K+-channel agonist properties may augment verapamil's vasodilatory effects. 4-Aminopyridine (4-AP) is a K+-channel antagonist. It has successfully reversed hypotension in experimental verapamil poisoning. We hypothesized that coadministration of these agents may improve BP and CO in verapamil poisoning. Anesthetized, ventilated, and canulated male Wistar rats were poisoned with verapamil. Animals received one of six treatments, which are as follows: (1) n-saline infusion (control), (2) Levo 6.25 µg/kg loading dose and 36 µg/kg/h infusion, (3) 4-AP 2 mg/kg loading dose and infusion (4-AP), (4) Levo+4-AP, (5) CaCl2 loading dose and infusion, and (6) Levo+CaCl(2). Hemodynamic parameters were recorded for 60 min. Outcome measures were changes in CO, BP, and heart rate (HR) compared to control. All groups had similar pretoxicity and peak toxicity CO (50% of pretoxicity value), BP (50% or pretoxicity value), and HR. Control group CO, BP, and HR progressively dropped during the verapamil infusion. Levosimendan produced a statistically significant improvement in CO (75% of pretoxicity level) but not BP in comparison to control. 4-AP produced a significant improvement in CO (110% of pretoxicity) and BP (78% of pretoxicity). Levo+4-AP and Levo+CaCl2 groups improved CO (100% of pretoxicity) and BP (77% and 50% of pretoxicity, respectively), but there was no additive increase in CO or BP in animals compared to 4-AP or CaCl2 alone. Levosimendan moderately improved CO but not BP in verapamil poisoning. The hypotensive effects of levosimendan were not overcome by coadministration of either 4-AP or CaCl2. Levosimendan may not be an appropriate agent to use in the treatment of verapamil poisoning.